The overall goal of this project is to identify the ion transport mechanisms which drive secretion of electrolytes in the acinus of the rat parotid gland. Because it is difficult to use traditional biophysical techniques in work on a tubuloacinar epithelium we will develop a new technical approach. That is, we will devise methods for isolating acinar cell apical- and basal lateral plasma membranes, and we will study sodium and chloride transport in the purified membrane vesicles. We will use cholesterol and a variety of enzyme activities as provisional markers for the plasma membranes. Independent evidence has validated use of Na, K-ATPase as a basal lateral membrane marker. There is, as yet, no unique marker for the apical plasma membrane. However, in analyzing the distributions of a variety of markers we should detect two populations of plasma membrane derived-particles, one rich in Na, K-ATPase and one lacking this activity; preliminary results support this prediction. We will verify that these are derived from the plasma membranes by experiments with nonpermeating labeling reagents. Three different separation procedures will be utilized in design of the membrane isolation method: ultracentrifugation, free flow electrophoresis, and counter current distribution in aqueous polymer two phase systems. These procedures, as well as density perturbation with digitonin, will also be used as physical criteria for purity, corroborating results obtained with markers for other organelles. Having obtained preparations of isolated plasma membranes we will use tracer and membrane ultrafiltration methodologies to study sodium and chloride transport mechanisms. In particular, we will determine whether the basal lateral membranes contain a sodium-chloride cotransport system and whether the apical plasma membranes contain a conductive pathway for chloride. With both types of membranes we will estimate the relative permeabilities to sodium, potassium, and chloride, and we will test for Na/H, Cl/OH, and Cl/HCO3 exchange reactions.